Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling.

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat ß cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.

In vitro assessment of endocrine disrupting potential of naphthenic Acid fractions derived from oil sands-influenced water.

Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast androgen screen. The mixtures were characterized by high resolution mass spectrometry, (1)H nuclear magnetic resonance, and attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed. Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent antiandrogen or antiestrogens.

Synthesis and Evaluation of Thiazoloquinolinones with Linkers To Enable Targeting of CD38.

Several monoclonal antibodies and inhibitors targeting CD38, an ectoenzyme overexpressed on malignant plasma cells, have previously been discovered. Herein, we expand structure-activity relationships of reported small-molecule thiazoloquinolinones and show that several 4-cyclohexylamino analogues have potent binding affinity for CD38 using surface plasmon resonance. Moreover, active amine analogues could be acylated and functionalized with alkyne and fluorescein groups. Fluorescein analogue 21 bound selectively to CD38 overexpressing cells, demonstrating the potential utility of thiazoloquinolinones as small-molecule conjugates for the delivery of therapeutic and imaging agents.

Analgesic substances derived from natural products (natureceuticals).

From the first recorded accounts, over 7000 years ago, various forms of natural products have been utilized to treat pain disorders. Prototypical examples of such natural products are the opium poppy (Papaver soniferum) and the bark of the willow tree (Salix spp.). It was not until the 19th century when individual compounds were isolated from these substances and were determined to posses the desired effects. The known sources of these substances have been thoroughly investigated. Over the last several decades, more analgesic substances have been purified from natural products resulting in novel structural classes and mechanisms of actions. Plants and other natural products described in historical ethnobotanical and ethnopharmacological literature have become of more recent interest in drug discovery efforts. These manuscripts and reports are being utilized to aid in the identification of natural products that have been historically employed in the alleviation of pain. A large factor that has highlighted the importance of discovering novel compounds to treat pain has been in the fundamental understanding of the complex mechanisms of pain transmission in the nervous system. Nociceptive processing involves many receptor classes, enzymes and signaling pathways. The identification of novel classes of compounds from natural sources may lead to advancing the understanding of these underlying pharmacological mechanisms. With the potential of uncovering new compounds with idealistic pharmacological profiles (i.e., no side effects, no addictive potential), natural products still hold great promise for the future of drug discovery especially in the treatment of pain disorders and potentially drug addictions.

Synthesis of oxazocenones via gold(I)-catalyzed 8-endo-dig hydroalkoxylation of alkynamides.

Several benzoxazocenones have been found to exhibit novel cellular activities. In the present study, we report a gold(I)-catalyzed 8-endo-dig hydroalkoxylation reaction of alkynamides to access analogous oxazocenone scaffolds. This methodology provided an advanced intermediate, which was elaborated to a des-benzo analog of a bioactive benzoxazocenone.

Thiopurine pharmacogenomics: association of SNPs with clinical response and functional validation of candidate genes.

AIM: We investigated candidate genes associated with thiopurine metabolism and clinical response in childhood acute lymphoblastic leukemia. MATERIALS & METHODS: We performed genome-wide SNP association studies of 6-thioguanine and 6-mercaptopurine cytotoxicity using lymphoblastoid cell lines. We then genotyped the top SNPs associated with lymphoblastoid cell line cytotoxicity, together with tagSNPs for genes in the 'thiopurine pathway' (686 total SNPs), in DNA from 589 Caucasian UK ALL97 patients. Functional validation studies were performed by siRNA knockdown in cancer cell lines. RESULTS: SNPs in the thiopurine pathway genes ABCC4, ABCC5, IMPDH1, ITPA, SLC28A3 and XDH, and SNPs located within or near ATP6AP2, FRMD4B, GNG2, KCNMA1 and NME1, were associated with clinical response and measures of thiopurine metabolism. Functional validation showed shifts in cytotoxicity for these genes. CONCLUSION: The clinical response to thiopurines may be regulated by variation in known thiopurine pathway genes and additional novel genes outside of the thiopurine pathway.

Small-molecule inhibitors of cytokine-mediated STAT1 signal transduction in ß-cells with improved aqueous solubility.

We previously reported the discovery of BRD0476 (1), a small molecule generated by diversity-oriented synthesis that suppresses cytokine-induced ß-cell apoptosis. Herein, we report the synthesis and biological evaluation of 1 and analogues with improved aqueous solubility. By replacing naphthyl with quinoline moieties, we prepared active analogues with up to a 1400-fold increase in solubility from 1. In addition, we demonstrated that 1 and analogues inhibit STAT1 signal transduction induced by IFN-γ.

Studies toward the synthesis of the epoxykinamycin FL-120B': discovery of a decarbonylative photocyclization.

Photo-Friedel-Crafts acylation of a naphthoquinone was attempted in an effort to access a diazobenzofluorenone en route to the epoxykinamycin natural product FL-120B'. Photoirradiation of the naphthoquinone substrate which resulted in the unexpected formation of a tetracyclic naphthofuran via a decarbonylative photocyclization process is described.

Encapsulation of polyanilines into graphite oxide.

Herein we report on the intercalation of polyaniline, poly(2-ethylaniline), and poly(2-propylaniline) into graphite oxide. This was achieved by taking advantage of the exfoliation/reconstruction properties of the layered host. The resulting intercalates were characterized by powder X-ray diffraction and thermogravimetric analysis.